The goal of this study is to determine the nerve growth factor (NGF) signal transduction mechanism - the molecular mechanism by which binding of NGF to its receptor is converted into an intracellular signal that initiates the characteristic program of cellular responses. NGF and other neuronotrophic factors are of central importance in the ontogeny and maintenance of neurons. These studies will therefore be relevant to basic understanding of the cellular mechanisms underlying control of cell number, survival, growth and differentiation in the nervous system. Such understanding has potentially significant application to neuronal repair and regeneration in the amelioration of genetic and other neuro-degenerative disorders, trauma, and stroke. To the extent that NGF serves as an intracellular signal outside of the nervous system these studies will yield insight into the development and physiology of these tissues as well. It is also expected that these studies will be relevant to understanding the mechanisms of action of other neuronotrophic and growth factors and therefore to neoplasia, given the associations known to exist between oncogenes and growth factors. This proposal for study of NGF signal transduction consists of four projects which make use primarily, if not exclusively, of the NGF- responsive PC12 cell line and of the previously isolated non-NGF- responsive PC12 mutants (PC12nnr lines). The first project involves studies of the NGF receptor (NGFR) itself, focussing on regulation of its affinity, internalization and intracellular transport. The other three projects are all aimed at identifying and isolating NGF transduction-related proteins, other than the NGFR itself, by three different approaches. Each approach exploits a different aspect of the nature of such proteins, based on the results of previous studies of the NGFR and the mechanism of action of NGF. These projects will identify and isolate proteins involved in NGF signal transduction and produce immunological and molecular probes for them thereby allowing future studies of their function and expression. One project will be the biochemical isolation of NGFR-associated proteins, for which previous results have suggested key functional roles, exploiting their affinity for the NGFR. Following the isolation of such proteins, antibodies recognizing them will be prepared. The second project involves identification of transduction-related proteins by introducing DNA from NGF-responsive cells into PC12nnr cells using means that facilitate molecular cloning of those introduced genes that restore to PC12nnr cells the ability to respond to NGF. Additional PC12 cell mutants defective in responses to NGF will be isolated following proviral insertion mutagenesis. This tags the mutated genes with viral sequences, facilitating molecular cloning of the genes of interest. This project will identify genes involved in transduction or in the molecular processes underlying neuronal differentiation and neurite outgrowth. The products of the genes cloned by these means are proteins required for NGF responses. Antibodies recognizing these proteins can be produced from the translation products of the cloned genes. Third, transduction-related proteins will be assumed to possess particular enzymatic activities, such as GTP binding or tyrosine kinase. The identification, the function, and the role in NGF signal transduction of neuronal proteins possessing such activities will be explored in these studies.